Citation: Wang
LP, Chen BX, Sun Y, Chen JP, Huang S, Liu YZ. Celastrol inhibits migration,
proliferation and transforming growth factor-β2-induced
epithelial-mesenchymal transition in lens epithelial cells. Int J Ophthalmol
2019;12(10):1517-1523. DOI:10.18240/ijo.2019.10.01
·Basic
Research·
Celastrol
inhibits migration, proliferation and transforming growth factor-β2-induced epithelial-mesenchymal
transition in lens epithelial cells
Li-Ping Wang, Bao-Xin Chen, Yan Sun, Jie-Ping Chen, Shan
Huang, Yi-Zhi Liu
State Key
Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen
University, Guangzhou 510060, Guangdong Province, China
Correspondence
to: Shan Huang
and Yi-Zhi Liu. State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic
Center, Sun Yat-sen University, Guangzhou 510060, Guangdong Province, China.
huangshan@gzzoc.com; yizhi_liu@aliyun.com
Received:
Abstract
AIM: To investigate the mechanism of celastrol in inhibiting lens epithelial
cells (LECs) fibrosis, which is the pathological basis of cataract.
METHODS: Human LEC line SRA01/04 was treated with celastrol
and transforming growth factor-β2 (TGF-β2).
Wound-healing assay, proliferation assay, flow cytometry, real-time polymerase
chain reaction (PCR), Western blot and immunocytochemical staining were used to
detect the pathological changes of celastrol on LECs. Then, we cultured
Sprague-Dawley rat lens in medium as a semi-in vivo model to find the
function of celastrol further.
RESULTS: We found that celastrol inhibited the migration of
LECs, as well as proliferation (P<0.05). In addition, it induced the
G2/M phase arrest by cell cycle-related proteins (P<0.01). Moreover,
celastrol inhibited epithelial-mesenchymal transition (EMT) by the blockade of
TGF-β/Smad and Jagged/Notch
signaling pathways.
CONCLUSION: Our study demonstrates that celastrol could inhibit
TGF-β2-induced lens fibrosis and
raises the possibility that celastrol could be a potential novel drug in
prevention and treatment of fibrotic cataract.
KEYWORDS: lens; cataract; fibrosis;
transforming growth factor-β2; celastrol
DOI:10.18240/ijo.2019.10.01
Citation: Wang
LP, Chen BX, Sun Y, Chen JP, Huang S, Liu YZ. Celastrol inhibits migration,
proliferation and transforming growth factor-β2-induced
epithelial-mesenchymal transition in lens epithelial cells. Int J Ophthalmol
2019;12(10):1517-1523
INTRODUCTION
Cataract is
the world’s number one blinding eye disease[1]. At
present, the treatments of cataract are cataract phacoemulsification and Nd:YAG
laser capsulotomy. These surgeries are mature, but the complications couldn’t
be avoided among a large number of patients, which brings a heavy economic
burden to society[2]. Lens fibrotic disorder,
including anterior subcapsular cataract (ASC) and posterior capsule
opacification (PCO), is the main cause of cataract. This study intends to
explore the pathogenesis of fibrotic cataract and bring new directions for the
prevention and treatment of cataract.
ASC and PCO
are fibrotic cataract sharing a similar pathological process[3-4]. ASC is a primary cataract, which is caused by in
situ proliferation, abnormal fibrosis of lens epithelial cells (LECs)[5]. PCO is a common complication of cataract surgery. It
is caused by postoperative residual LECs migrating to the posterior capsule,
undergoing abnormal fibrosis. Then, the posterior capsule begins to shrink and
be cloudy[6]. Pathological epithelial-mesenchymal
transition (EMT) plays an important role in the development of fibrotic
cataract. During EMT process, LECs lose their original epithelial morphology,
gradually elongate to form mesenchymal-like cells and secrete extracellular
matrix (fibronectin, collagens IV). At the molecular level, the expression of
EMT marker (ɑ-smooth muscle actin) has also increased[4].
EMT is affected by many growth factors and signaling pathways. Transforming
growth factor-β2 (TGF-β2) is the main component in the aqueous humor and the
most important growth factor in the occurrence of EMT[7].
Therefore, suppressing the migration, proliferation and TGF-β2 induced EMT
maybe an effective measure.
Celastrol is
an active compound extracted from the roots of Tripterygium wilfordii. It has a
wide range of biological functions, including anti-oxidant, anti-inflammatory,
anti-tumor and immunomodulatory[8]. Recently,
studies have also found that celastrol can reduce pain and cartilage damage
caused by osteoarthritis[9]. Moreover, it can
protect myocardium from ischemia-reperfusion injury[10],
help improving learning and memory[11]. Many
studies have confirmed that celastrol can inhibit the proliferation, migration
and EMT of tumor cells[12-13].
For example, it can inhibit EMT of lung cancer cells through TGF-β/Smad
signaling pathway[14]. However, the research of
celastrol on the eye is mainly confined to the retina. It can protect the
retinal ganglion cell from degeneration caused by high intraocular pressure[15]. Also, it is helpful to the treatment of optic
neuritis and retina experiencing light damage by inhibiting oxidative stress
and inflammation[16]. Moreover, celastrol can
regulate the innate immune response of retinal pigment epithelial cells through
the NF-κB and Hsp70[17]. However, the role of
celastrol in lens fibrosis is still unclear. This study first confirms that
celastrol could inhibit the fibrosis of LECs induced by TGF-β2. It can
significantly inhibit the migration and proliferation of LECs and cause cell
cycle arrest. Moreover, EMT in LECs is inhibited by inactivation of the
classical TGF-β2/Smad. Also, celastrol could regulate the Jagged/Notch pathways.
In summary, this study demonstrates that celastrol could be a new drug for the
treatment of fibrotic cataract in the future.
MATERIALS AND METHODS
Reagents and
Antibodies Celastrol (≥98%) was purchased from
Sigma-Aldrich. Recombinant human TGF-β2 was purchased from R&D SYSTEMS.
Antibodies against cdc2 (polyclonal), cycling B1 (monoclonal), p-Smad2/3
(monoclonal), Smad2/3 (polyclonal), Notch1 (monoclonal), Notch3(monoclonal),
Jagged1(monoclonal), GAPDH (monoclonal) were purchased from Cell Signaling
Technology. α-SMA (polyclonal), Col IV (polyclonal) and FN (polyclonal) were
purchased from Abcam.
Cell Culture
and Treatment The human LEC line SRA01/04 was
provided by professor Fu Shang from Nutrition and Vision Research Laboratory,
Tufts University (Boston, MA, USA). They were cultured in Dulbecco’s modified
Eagle’s medium (DMEM, Gibco, Life Technologies, NY, USA) containing 10% fetal
bovine serum (FBS, Gibco, Life Technologies) at
Wound-Healing
Assay LECs were seeded in the six-well
plate. When the cell density of each well reached 90% confluence, cells were
starved by DMEM containing 1% FBS overnight. Then LECs were scratched with a
200 µL pipette tip. Wounded monolayers were washed with phosphate buffer saline
(PBS) to remove detached cells, and fresh medium was added to each well. The
wound in each well was photographed at 0, 4, 8 and 12h. The length of the
remaining wound in each image was measured 3 times using Zeiss software.
Cell
Proliferation Assay LECs were seeded in the 96-well
plate in triplicate. When the cell density of each well reached 50% confluence,
we pipeted 20 µL CellTiter 96® AQueous One Solution
Reagent (Promega) into each well containing 100 µL samples and incubated the
plate at
Cell Cycle
Analysis Cells were treated with increasing
concentrations of celastrol for 24h. After harvested and fixed in cold 70%
ethanol overnight at
Western Blot
Analysis Cells were lysed in radio
immunopreci pitation assay (RIPA) buffer and protein was mixed with 5×SDS
sample buffer. An equal amount of total protein from each group was separated
by 10% sodium dodecylsulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and
transferred to polyvinylidene fluoride (PVDF) membranes. The membranes were
blocked with 5% nonfat milk for 1h at room temperature and incubated overnight
at
Real-time
Polymerase Chain Reaction Analysis Total RNA was isolated from LECs
according to the manufacturer’s protocol (QIAGEN). cDNA was synthesized with
reverse transcription kit (Thermo Scientific). For real-time polymerase chain
reaction (PCR), the SYBR (Roche) was used according to the manufacturer’s
protocol with the ABI Prism 7000 sequence detection system (Applied Biosystems,
Foster City, CA, USA).
Immunofluorescence
LECs were fixed by 4%
paraformaldehyde and permeabilized with 0.3% Triton X
Lens Culture
and Treatment Animals included in this study were
approved by the Animal Use and Care Committee of Zhongshan Ophthalmic Center
(Guang Zhou, People’s Republic of China) and has been performed in accordance
with the ARVO statement. Lens from
Statistical
Analysis Each experiment was repeated at
least three times. All data were expressed as mean±SD and analyzed with SPSS
15.0 software (SPSS Inc., Chicago, IL, USA). Two tailed Student’s t-test
was used to calculate the P value between the groups. A value of P<0.05
was considered statistically significant.
RESULTS
Celastrol
Inhibited the Migration and Proliferation of LECs The migration and proliferation of
residual LECs is the original cause of PCO. To evaluate the effect of celastrol
on the migration of LECs, we performed wound-healing assay. The distance of
cell migration was observed by an inverted phase contrast microscope at 0, 4, 8
and 12h (Figure
Figure 1
Celastrol inhibited the migration and proliferation of LECs A: LECs were treated with or without
celastrol (1 μmol/L) for 12h. Pictures were captured every 4h and the black
lines represented the wound edges (scale bar 100 μm); B: The distance of LECs
migration was measured at different time points; C: LECs were treated with 0,
0.5, 1 μmol/L celastrol for 0, 1, 2, 3, 4d. Data were the mean±SD from an
experiment which was repeated three times. aP<0.05 vs
the control group.
Celastrol
Induced G2/M Phase Arrest by Regulating Cell Cycle-related Proteins Cell cycle progression has influence
on cell proliferation. Then, we further examined the effect of celastrol on
cell cycle. Distribution of cell cycle in LECs treated with different
concentration of celastrol was detected. The percentages of LECs in G2 phase
were 14.8%±3.1%, 19.6%±3.6%, 27.6%±2.1% for the control, 1, 2 μmol/L groups (P<0.01
vs the control group; Figure
Figure 2
Celastrol induced G2/M phase arrest by cell cycle-regulated proteins A, B: LECs were treated with 0, 1, 2
μmol/L celastrol for 24h. Cell cycle was detected by flow cytometry. The
percentage of LECs in G2 phase was calculated; C: LECs were treated with 0,
0.5, 1, 1.5, 2 μmol/L celastrol for 48h. The protein expression levels of cdc2
and cyclin B1 were detected by Western blot analysis. D: Quantitative analysis
of protein levels were detected
three times. Data were derived by three independent experiments. bP<0.01
vs the control group.
Celastrol
Inhibited TGF-β2-induced EMT in LECs by Suppressing the Phosphorylation of
Smad2/3 TGF-β2 is crucial in the formation
of fibrotic cataract[4]. To detect the role of
celastrol in LECs EMT, we used TGF-β2 to establish an in vitro model.
TGF-β2 increased the expression of FN and Col IV at mRNA levels. On the contrary,
celastrol abrogated their upregulation (P<0.05 vs the TGF-β2
group; Figure
Figure 3
Celastrol inhibited TGF-β2-induced EMT in LECs LECs were cultured in the absence or
presence of TGF-β2 (5 ng/mL) with celastrol (1 μmol/L) for 24-48h. A: The mRNA
expression level of FN and Col IV in LECs was detected by real-time PCR; B, C:
The protein expression level of FN was determined by Western blot analysis; D:
Immunofluorescence analysis of FN (green), Col IV (green) and α-SMA (red) were
observed using confocal microscopy (scale bar 50 μm). Data were derived by
three independent experiments. aP<0.05, bP<0.01
vs the TGF-β2 group.
Celastrol
Regulated the Jagged/Notch Signaling Pathway in LECs The Jagged/Notch signaling pathway
plays an important role in embryonic development, fibrotic diseases and cancer
metastasis[19]. To investigate the effect of
celastrol on LECs fibrosis further, we explored the impact of celastrol on the
Jagged/Notch signaling pathway. The TGF-β2 group significantly increased the
expression of Notch2, Notch3, Jagged1 at mRNA levels (Figure
Figure 4
Celastrol inhibited TGF-β2 signaling pathway by suppressing the phosphorylation
of Smad2/3 and downregulating the Jagged/Notch signaling pathway LECs were cultured in the absence or
presence of TGF-β2 (5 ng/mL) with celastrol (1 μmol/L) for 24-48h. A: The mRNA
expression levels of Notch2, Notch3 and Jagged
Celastrol
Suppressed TGF-β2-induced ASC To further investigate whether
celastrol could prevent TGF-β2-induced fibrosis in lens, we cultured the whole
lens in semi-in vivo model. Lens had obvious anterior opacity treated
with TGF-β2 (Figure
Figure 5
Celastrol suppressed TGF-β2-induced ASC in the whole lens culture semi-in
vivo mode LECs were cultured in the absence or
prescence of TGF-β2 (5 ng/mL) with celastrol (1 μmol/L) for a week. A: The
morphology of lens was captured by dissecting microscope (scale bar 500 μm); B:
The staining of parafin section of α-SMA (red) was captured by confocal
microscopy (scale bar 100 μm).
DISCUSSION
Celastrol is
a natural product with a variety of biological activities. It can inhibit the
EMT of many tumor cells[20], but its role in
fibrotic cataract is not clear. Fibrotic cataract shows an increase in cell
migration, proliferation and pathological changes of EMT[21].
Therefore, we explore from these aspects to find the pathomechanisms of fibrotic
cataract. In this study, we first discovered that celastrol could inhibit
TGF-β2-induced LECs fibrosis. We demonstrated that celastrol could 1) inhibit
the migration and proliferation of LECs; 2) cause G2/M phase arrest; 3) inhibit
the process of EMT by inactivating Smad2/3 phosphorylation; 4) regulate the
Jagged/Notch signaling pathway.
During the
process of EMT, migratory mesenchymal cell types were produced, which have the
ability of enhanced motility and proliferation[22-23]. Increased LECs migration and proliferation is the
primary cause of fibrotic cataract. There are many factors that affect cell
proliferation, and cell cycle arrest has a close relationship with it[24]. The progression of cell cycle depends on the
coordination of cell cycle-related proteins and signaling pathways, which
ultimately affect cell proliferation[25]. For
example, cdc2 binds to cyclin B. The complex promotes cell cycle progression
from G2 to M[26]. Celastrol can cause G2/M arrest
in a variety of tumor cells[27-28],
but its function on LECs is unclear. We found that it could reduce the expression
of cyclin B1 and cdc2. Therefore, this may be another mechanism of celastrol
inhibiting the proliferation of LECs.
EMT plays an
important role in LECs fibrosis. TGF-β2 signaling pathway is crucial in many
fibrotic diseases. It can activate phosphorylation of Smad2/3[29]. At present, more and more studies have found that
the Jagged/Notch signaling pathway plays an important role in fibrotic
diseases, such as fibrotic cataract[30-31].
We found that the phosphorylation of Smad2/3 was increased when LECs were
induced by TGF-β2, but the phosphorylation level was decreased treated with
celastrol. In addition, it could inhibit the Jagged/Notch signaling pathway.
During the development of EMT, there is a change in related markers, such as
FN, Col IV and α-SMA. We found celastrol reduced the expression of FN, Col IV
and α-SMA induced by TGF-β2. Therefore, we confirm that celastrol could inhibit
the EMT of LECs.
The lens
consists of LECs, cortex and capsular membranes. It is a good model for
studying LECs. Many studies have shown that TGF-β2 induces lens to undergo
morphological and molecular changes which are similar to fibrotic cataract[32]. In order to verify the function of celastrol on lens
fibrosis, we cultured the whole Sprague-Dawley rat lens with TGF-β2 to simulate
the in vivo environment. We found that lens treated with celastrol was
more transparent and the expression of ɑ-SMA was decreased. Through these
results, we find that celastrol can keep the transparency of lens and inhibit
the formation of cataract.
In summary,
our study confirms that celastrol can inhibit the fibrosis of LECs for the
first time. Celastrol not only inhibits the migration and proliferation of
LECs, but also leads to cell cycle arrest. At the same time, celastrol inhibits
EMT by inactivating TGF-β/Smad and regulate the Jagged/Notch signaling
pathways. Our study provides a new direction for the prevention and treatment
of fibrotic cataract, and it could benefit a majority of cataract patients in
the future.
ACKNOWLEDGEMENTS
Foundations: Supported by National Natural
Science Foundation of China (No.81300749); Guangdong Province Natural Science
Foundation (No
Conflicts of
Interest: Wang LP, None; Chen BX, None; Sun Y, None; Chen JP, None;
Huang S, None; Liu YZ, None.
REFERENCES